Cyclohexyldimethylammonium dihydrogen phosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

July 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Mar 2006 (corrected Jul 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest test solution of nominal 100 mg/L was prepared in culture medium. From this solution a series of dilutions was made to give further solutions of 32, 10, 3.2 and 1.0 mg/L. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
- Controls: test water

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Ohan, Argyll, Scotland.
- Age of inoculum (at test initiation): not reported. Prior to the start of the test master culture was added to culture media to give an initial cell density of approximately 10E+03 cells/mL. The flasks were kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E+04 - 10E+05 cells/mL.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium under constant aeration and constant illumination at 21 ± 1 °C.

ACCLIMATION
- Acclimation period: see above "Age of inoculum"
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg CaCO3/L

Test temperature:

24 ± 1 °C

pH:

control
test start (0 h): 7.0
test end (72 h): 7.1

treatments
test start (0 h): 6.6 - 7.1
test end (72 h): 6.9 - 7.5

the lower pH values in the treatments were observed at the higher test item concentrations

Nominal and measured concentrations:

nominal concentrations: 1.0, 3.2, 10, 32, and 100 mg/L
measured concentrations:
start: 94 - 96 % of nominal
end (72 h): 93 - 97 % of nominal
The biological results are based on the nominal concentration of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks containing 100 mL test medium, plugged with polyurethane foam bungs
- Aeration: continuous shaking at about 150 rpm
Initial cells density: 5 x 10E+03 cells/mL
- Control end cells density: 1.07 x 10E+06 cells/mL

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured in each treatment at the start and end of the test. The temperature in the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: approximately 7000 lux, warm white light (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): daily determination of the algal cell density
- Determination of cell concentrations: electronic particle counter; triplicate counts


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 76 % inhibition of growth rate at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval: 23 - 35 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 7 - 8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: values were read off the growth curve

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities in the control and at 1.0 and 3.2 mg/L; cell debris was observed at 10 mg/L and no intact cells were observed in the 32 and 100 mg/L test cultures.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: none

For details see attached document "Tables Cell densities and inhibition of growth.pdf"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hour ErC50: 1.2 mg/L, 95% confidence limits 1.1 - 1.4 mg/L (within the range 0.9 - 1.5 mg/L as recommended by the guidelines)

Reported statistics and error estimates:

Statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups.

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, (OECD 201, EU C.3):

EC50: 28 mg/L, EC10: 7 - 8 mg/L, NOEC: 3.2 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

28 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

EC10 for freshwater algae: 7 - 8 mg/L